Preventing endogenous aminopeptidase mediated n-terminal amino acid cleavage during expression of foreign genes in bacteria

Abstract

A process for the recombinant preparation in a bacterial host of the mature form of a mammalian protein or peptide of formula X-Pro-Z, which is subject to processing by endogenous bacterial aminopeptidases is provided. In the formula X-Pro-Z, X is a single N-terminal amino acid other than proline, and Z is the remaining sequence of amino acid residues of the protein or peptide. The process comprises inserting into a cell of the bacterial host an appropriate vector containing DNA coding for Met-Y-X-Pro-Z, in which Y is a natural amino acid that is specifically cleavable in vitro from X-Pro-Z by an aminopeptidases and that imparts resistance to in vivo processing by endogenous bacterial aminopeptidases. The cell is then induced to express the expression product Met-Y-X-Pro-Z which is then treated with an appropriate aminopeptidase to cleave off Met and Y.

What is claimed is: 
     
       1. A process for the recombinant production in a bacterial host of the mature form of a mammalian protein which is subject to processing by endogenous bacterial aminopeptidases of the formula X-Pro-Z, comprising the steps of: a) transforming bacterial host cells with an appropriate vector containing DNA coding for Met-Y-X-Pro-Z (SEQ. ID NO. 1);   b) inducing the cell to express the Met-Y-X-Pro-Z product;   c) treating the Met-Y-X-Pro-Z product with an appropriate aminopeptidase to cleave off Met and Y; and   d) isolating the resulting protein X-Pro-Z, wherein X-Pro-Z is human interleukin 6 (IL-6) or leukemia inhibitory factor (LIF), X is a single N-terminal amino acid other than proline, Z is the remaining sequence of amino acid residues of the protein, and Y is a natural amino acid selected from Ser, Asn, Gln, His, Arg, Lys, Tyr, Phe, Met, Val, Ile and Leu which is specifically cleavable in vitro from Y-X-Pro-Z (SEQ. ID NO. 2) by an aminopeptidase and imparts resistance to in vivo processing by endogenous bacterial aminopeptidases.     
     
     
       2. A process for the production of a precursor of a mature mammalian protein product of sequence X-Pro-Z, which comprises expressing the precursor in bacterial host cells, the precursor having the sequence Met-Y-X-Pro-Z (SEQ. ID NO. 1), wherein X-Pro-Z is IL-6 or LIF, Y is a natural amino acid selected from Ser, Asn, Gln, His, Arg, Lys, Tyr, Phe, Met, Val, Ile and Leu which is specifically cleavable in vitro from Y-X-Pro-Z (SEQ. ID NO. 2) by an aminopeptidase and impart resistance to in vivo processing by endogenous bacterial aminopeptidases, X is a single N-terminal amino acid other than proline, and Z is the remaining amino acid sequence of the mature product. 
     
     
       3. A process for the production of a mature mammalian protein product of sequence X-Pro-Z, which comprises treating a precursor which has the sequence Met-Y-X-Pro-Z (SEQ. ID NO. 1) with an aminopeptidase to yield the mature product, wherein X-Pro-Z is IL6 or LIF, X is a single N-terminal amino acid other than proline, Z is the remaining amino acid sequence of the mature product, and Y is a natural amino acid selected from Ser, Asn, Gln, His, Arg, Lys, Tyr, Phe, Met, Val, Ile and Leu which is specifically cleavable in vitro from Y-X-Pro-Z (SEQ. ID NO. 2) by an aminopeptidase and imparts resistance to in vivo processing by endogenous bacterial aminopeptidases. 
     
     
       4. A process according to claim 1, in which the protein X-Pro-Z is IL-6 and Y is Phe, His, Met, Ile or Leu. 
     
     
       5. A process according to claim 4 in which Y is Phe, His or Met. 
     
     
       6. A process according to claim 1 in which the protein X-Pro-Z is LIF and Y is His, Phe or Ile. 
     
     
       7. A process according to claim 6 in which Y is Ile. 
     
     
       8. A process according to claim 1 in which the bacterial host is E. coli. 
     
     
       9. A process according to claim 1 in which the aminopeptidase used to cleave off Met and Y is selected from those obtained from Bacillus stearothermophilus, Streptomyces griseus or Aeromonas. 
     
     
       10. A process according to claim 9 in which the aminopeptidase is obtained from Aeromonas. 
     
     
       11. An N-terminal extended precursor of a mature mammalian protein product having the sequence Met-Y-X-Pro-Z (SEQ. ID NO. 1) or Y-X-Pro-Z (SEQ. ID NO. 2), wherein X-Pro-Z is IL-6 or LIF, X is a single amino acid other than proline, Z is the remaining amino acid sequence of the mature product, and Y is a natural amino acid selected from Ser, Asn, Gln, His, Arg, Lys, Tyr, Phe, Met, Val, Ile and Leu which is specifically cleavable in vitro from Y-X- Pro-Z (SEQ. ID NO. 2) by an aninopeptidase and imparts resistance to in vivo processing by endogenous bacterial aminopeptidases. 
     
     
       12. A precursor according to claim 11 in which X-Pro-Z is IL-6 and Y is Phe, His, Met, Ile or Leu. 
     
     
       13. A precursor according to claim 11 in which X-Pro-Z is LIF and Y is His, Phe or Ile.